Monoclonal Antibody Sc104 and Derivative Thereof Specifically Binding to a Sialyltetraosyl Carbohydrate as a Potential Anti-Tumor Therapeutic Agent

ABSTRACT

The present invention provides an isolated specific binding member capable of binding a sialyltetraosly carbohydrate and directly inducing cell death without the need for immune effector cells. Such a binding member may be an antibody or a part thereof. Also provided are the use of such binding members in medicine and nucleic acids encoding such binding members.

The present invention relates to specific binding members, particularlyantibodies and fragments thereof, which bind to an epitope relevant tocancer and endometriosis. Such members are useful for the diagnosis andtreatment of tumours.

Antibodies which bind to GM2, GD3 and GM3 gangliosides are known, but anantibody that binds a sialyltetraosylceramide, but which does not bindto GM1, GD1a, GT1b or sialyl Lewis antigens has not previously beendescribed. A mouse monoclonal antibody (mab), known herein as “SC104”,was raised to sequential immunisation with 4 colorectal cancer celllines and binds to 71% of colorectal tumours. SC104 is specific for asialyltetraosyl carbohydrate, which may be present on a lipid (ceramide)or protein backbone, and recognises oesophageal, colorectal, gastric,breast, parotid and endometrial tumours. SC104 is unusual as it is anIgG1 antibody. One of the immunological characteristics of carbohydrateantigens is that they usually elicit a T-cell independent response,resulting in the production of an IgM antibody. SC104 was shown byimmunostaining HPLTC plates of lipid extracts from the colorectal cellline C170, to bind to a sialyltetraosylceramide. SC104 was also shown tobind to a protein moiety which has the sialyltetraosyl carbohydrate.Recognition of normal tissue was minimal and restricted to moderatestaining of the large intestine, salivary gland, small intestine,thymus, tonsils and uterine cervix. The present inventors also,surprisingly, found that the antibody induced cell death.

According to a first aspect of the present invention, there is providedan isolated specific binding member capable of binding a sialyltetraosylcarbohydrate and of directly inducing cell death without the need forimmune effector cells.

A second aspect of the invention provides an isolated specific bindingmember capable of binding a sialyltetraosyl carbohydrate, whichsialyltetraosyl carbohydrate is capable of being bound by a membercomprising one or more binding domains selected from domains comprisingan amino acid sequence substantially as set out as residues 44 or 49 to54, 69 to 84 and 117 to 127 of the amino acid sequence of FIG. 1 a.

The binding domain may comprise an amino acid sequence substantially asset out as residues 117 to 127 of FIG. 1 a.

In one embodiment, the isolated specific binding member of the secondaspect of the present invention comprises one or more binding domainsselected from domains which comprise an amino acid sequencesubstantially as set out as residues 44 or 49 to 54, 69 to 84 or 117 to127 of the amino acid sequence of FIG. 1 a.

In one embodiment, the member comprises a binding domain which comprisesan amino acid sequence substantially as set out as residues 117 to 127of the amino acid sequence of FIG. 1 a. In this embodiment, the isolatedspecific binding member may additionally comprise one or both,preferably both, of the binding domains substantially as set out asresidues 44 or 49 to 54 and residues 69 to 84 of the amino acid sequenceshown in FIG. 1 a.

One isolated specific binding member of the second aspect of theinvention comprises the amino acid sequence substantially as set out asresidues 19 to 138 of the amino acid sequence shown in FIG. 1 a.

In a third aspect, the present invention provides an isolated specificbinding member capable of binding a sialyltetraosyl carbohydrate, whichsialyltetraosyl carbohydrate is capable of being bound by a membercomprising one or more binding domains selected from domains comprisingan amino acid sequence substantially as set out as residues 46 to 55, 71to 77 and 110 to 118 of the amino acid sequence of FIG. 1 c.

The binding domain may comprise an amino acid sequence substantially asset out as residues 110 to 118 of the amino acid sequence of FIG. 1 c.

In one embodiment, the isolated specific binding member of the thirdaspect of the present invention comprises one or more binding domainsselected from domains which comprise an amino acid sequencesubstantially as set out as residues 46 to 55, 71 to 77 and 110 to 118of the amino acid sequence of FIG. 1 c.

In one embodiment, the member comprises a binding domain which comprisesan amino acid sequence substantially as set out as residues 110 to 118of the amino acid sequence of FIG. 1 c. In this embodiment, the isolatedspecific binding member may additionally comprise one or both,preferably both, of the binding domains substantially as set out asresidues 46 to 55 and residues 71 to 77 of the amino acid sequence shownin FIG. 1 c.

Specific binding members which comprise a plurality of binding domainsof the same or different sequence, or combinations thereof, are includedwithin the present invention. The or each binding domain may be carriedby a human antibody framework. For example, one or more binding regionsmay be substituted for the CDRs of a whole human antibody or of thevariable region thereof.

One isolated specific binding member of the third aspect of theinvention comprises the sequence substantially as set out as residues 23to 128 of the amino acid sequence shown in FIG. 1 c.

In a fourth aspect, the invention provides a specific binding memberwhich comprises a binding member of the second aspect in combination orassociation with a binding member of the third aspect. Such a bindingmember may be in the form of a F_(v), (Fab′)₂, or scFV antibodyfragment.

Specific binding members of the invention may carry a detectable orfunctional label.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a specific binding member of thefirst, second, third or fourth aspects of the invention, and methods ofpreparing specific binding members of the invention which compriseexpressing said nucleic acids under conditions to bring about expressionof said binding member, and recovering the binding member.

Specific binding members according to the invention may be used in amethod of treatment or diagnosis of the human or animal body, such as amethod of treatment of a tumour in a patient (preferably human) whichcomprises administering to said patient an effective amount of aspecific binding member of the invention. The invention also provides aspecific binding member of the present invention for use in medicine, aswell as the use of a specific binding member of the present invention inthe manufacture of a medicament for the diagnosis or treatment of atumour.

The invention also provides the antigen to which the specific bindingmembers of the present invention bind. In one embodiment, asialyltetraosyl carbohydrate which is capable of being bound, preferablyspecifically, by a specific binding member of the present invention isprovided. The sialyltetraosyl carbohydrate may be provided in isolatedform, and may be used in a screen to develop further specific bindingmembers therefor. For example, a library of compounds may be screenedfor members of the library which bind specifically to thesialyltetraosyl carbohydrate. The sialyltetraosyl carbohydrate may on alipid backbone (i.e. a sialyltetraosylceramide) or on a proteinbackbone. When on a protein backbone, it may have a molecular weight ofabout 50-75 kDa, as determined by SDS-PAGE.

These and other aspects of the invention are described in further detailbelow.

As used herein, a “specific binding member” is a member of a pair ofmolecules which have binding specificity for one another. The members ofa specific binding pair may be naturally derived or wholly or partiallysynthetically produced. One member of the pair of molecules has an areaon its surface, which may be a protrusion or a cavity, whichspecifically binds to and is therefore complementary to a particularspatial and polar organisation of the other member of the pair ofmolecules. Thus, the members of the pair have the property of bindingspecifically to each other. Examples of types of specific binding pairsare antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is generallyconcerned with antigen-antibody type reactions, although it alsoconcerns small molecules which bind to the antigen defined herein.

As used herein, “treatment” includes any regime that can benefit a humanor non-human animal, preferably mammal. The treatment may be in respectof an existing condition or may be prophylactic (preventativetreatment).

As used herein, a “tumour” is an abnormal growth of tissue. It may belocalised (benign) or invade nearby tissues (malignant) or distanttissues (metastatic). Tumours include neoplastic growths which causecancer and include oesophageal, colorectal, gastric, breast andendometrial tumours, as well as cancerous tissues or cell linesincluding, but not limited to, leukaemic cells. As used herein, “tumour”also includes within its scope endometriosis.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen, whether natural or partly or wholly synthetically produced.The term also covers any polypeptide or protein having a binding domainwhich is, or is homologous to, an antibody binding domain. These can bederived from natural sources, or they may be partly or whollysynthetically produced. Examples of antibodies are the immunoglobulinisotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypicsubclasses; fragments which comprise an antigen binding domain such asFab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal ormonoclonal. A monoclonal antibody may be referred to herein as “mab”.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementary determining regions(CDRs), of an antibody to the constant regions, or constant regions plusframework regions, of a different immunoglobulin. See, for instance,EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or other cellproducing an antibody may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding member or substancehaving a binding domain with the required specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, humanized antibodies, including anypolypeptide comprising an immunoglobulin binding domain, whether naturalor wholly or partially synthetic. Chimeric molecules comprising animmunoglobulin binding domain, or equivalent, fused to anotherpolypeptide are therefore included. Cloning and expression of chimericantibodies are described in EP-A-0120694 and EP-A-0125023. A humanizedantibody may be a modified antibody having the variable regions of anon-human, e.g. murine, antibody and the constant region of a humanantibody. Methods for making humanized antibodies are described in, forexample, U.S. Pat. No. 5,225,539

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341:544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., Science 242:423-426 (1988); Huston etal., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

Diabodies are multimers of polypeptides, each polypeptide comprising afirst domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (e.g. by apeptide linker) but unable to associated with each other to form anantigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Hollinger & Winter, Current Opinion Biotechnol. 4:446-449 (1993)), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. It may be preferable touse scFv dimers or diabodies rather than whole antibodies. Diabodies andscFv can be constructed without an Fc region, using only variabledomains, potentially reducing the effects of anti-idiotypic reaction.Other forms of bispecific antibodies include the single chain “Janusins”described in Traunecker et al., EMBO Journal 10:3655-3659 (1991).

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be useful because they can be readily constructed and expressed inE. coli. Diabodies (and many other polypeptides such as antibodyfragments) of appropriate binding specificities can be readily selectedusing phage display (WO94/13804) from libraries. If one arm of thediabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.

An “antigen binding domain” is the part of an antibody which comprisesthe area which specifically binds to and is complementary to part or allof an antigen. Where an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by one or more antibody variabledomains. An antigen binding domain may comprise an antibody light chainvariable region (VL) and an antibody heavy chain variable region (VH).

“Specific” is generally used to refer to the situation in which onemember of a specific binding pair will not show any significant bindingto molecules other than its specific binding partner(s), and, e.g., hasless than about 30%, preferably 20%, 10%, or 1% cross-reactivity withany other molecule. The term is also applicable where e.g. an antigenbinding domain is specific for a particular epitope which is carried bya number of antigens, in which case, the specific binding membercarrying the antigen binding domain will be able to bind to the variousantigens carrying the epitope.

“Isolated” refers to the state in which specific binding members of theinvention or nucleic acid encoding such binding members will preferablybe, in accordance with the present invention. Members and nucleic acidwill generally be free or substantially free of material with which theyare naturally associated such as other polypeptides or nucleic acidswith which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practiced in vitro or invivo. Specific binding members and nucleic acid may be formulated withdiluents or adjuvants and still for practical purposes be isolated—forexample, the members will normally be mixed with gelatin or othercarriers if used to coat microtitre plates for use in immunoassays, orwill be mixed with pharmaceutically acceptable carriers or diluents whenused in diagnosis or therapy. Specific binding members may beglycosylated, either naturally or by systems of heterologous eukaryoticcells, or they may be (for example if produced by expression in aprokaryotic cell) unglycosylated.

By “substantially as set out” it is meant that the CDR regions of theinvention will be either identical or highly homologous to the specifiedregions of FIGS. 1 a and 1 c. By “highly homologous” it is contemplatedthat from 1 to 5, from 1 to 4, from 1 to 3, 2 or 1 substitutions may bemade in the CDRs.

The invention also includes within its scope polypeptides having theamino acid sequence as set out in FIG. 1 a or 1 c, polynucleotideshaving the nucleic acid sequences as set out in FIG. 1 a or 1 c andsequences having substantial identity thereto, for example, 70%, 80%,85%, 90%, 95% or 99% identity thereto. The percent identity of two aminoacid sequences or of two nucleic acid sequences is generally determinedby aligning the sequences for optimal comparison purposes (e.g., gapscan be introduced in the first sequence for best alignment with thesecond sequence) and comparing the amino acid residues or nucleotides atcorresponding positions. The “best alignment” is an alignment of twosequences that results in the highest percent identity. The percentidentity is determined by comparing the number of identical amino acidresidues or nucleotides within the sequences (i.e., % identity=number ofidentical positions/total number of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search that detects distant relationshipsbetween molecules (Id.). When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the GCG sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

Isolated specific binding members of the present invention are capableof binding to a sialyltetraosyl carbohydrate, which may be asialyltetraosylceramide or may be on a protein moiety. In oneembodiment, the CDR3 regions, comprising the amino acid sequencessubstantially as set out as residues 117 to 127 of FIG. 1 a and 110 to118 of FIG. 1 c, are carried in a structure which allows the binding ofthese regions to a sialyltetraosyl carbohydrate.

The structure for carrying the CDR3s of the invention will generally beof an antibody heavy or light chain sequence or substantial portionthereof in which the CDR3 regions are located at locations correspondingto the CDR3 region of naturally-occurring VH and VL antibody variabledomains encoded by rearranged immunoglobulin genes. The structures andlocations of immunoglobulin variable domains may be determined byreference to Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 4^(th) Edition, US Department of Health andHuman Services, (1987), and updates thereof, now available on theInternet (http://immuno.bme.nwu/edu)).

The amino acid sequence substantially as set out as residues 117 to 127of FIG. 1 a may be carried as the CDR3 in a human heavy chain variabledomain or a substantial portion thereof, and the amino acid sequencesubstantially as set out as residues and 110 to 118 of FIG. 1 c may becarried as the CDR3 in a human light chain variable domain or asubstantial portion thereof.

The variable domains may be derived from any germline or rearrangedhuman variable domain, or may be a synthetic variable domain based onconsensus sequences of known human variable domains. The CDR3-derivedsequences of the invention may be introduced into a repertoire ofvariable domains lacking CDR3 regions, using recombinant DNA technology.

For example, Marks et al (Bio/Technology 10:779-783 (1992)) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of WO92/01047 so that suitable specific bindingmembers may be selected. A repertoire may consist of from anything from10⁴ individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰members.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature 370:389-391 (1994)) who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying theCDR3-derived sequences of the invention using random mutagenesis of, forexample, the SC104 VH or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al(Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992)), who used error-pronePCR.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al (Proc.Natl. Acad. Sci. USA 91:3809-3813 (1994)) and Schier et al (J. Mol.Biol. 263:551-567 (1996)).

A substantial portion of an immunoglobulin variable domain willgenerally comprise at least the three CDR regions, together with theirintervening framework regions. The portion may also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps,including the introduction of linkers to join variable domains of theinvention to further protein sequences including immunoglobulin heavychains, other variable domains (for example in the production ofdiabodies) or protein labels as discussed in more detail below.

One embodiment of the invention provides specific binding memberscomprising a pair of binding domains based on the amino acid sequencesfor the VL and VH regions substantially as set out in FIGS. 1 a and 1 c,i.e. amino acids 19 to 138 of FIG. 1 a and amino acids 23 to 128 of FIG.1 c. Single binding domains based on either of these sequences formfurther aspects of the invention. In the case of the binding domainsbased on the amino acid sequence for the VH region substantially set outin FIG. 1 a, such binding domains may be used as targeting agents sinceit is known that immunoglobulin VH domains are capable of binding targetantigens in a specific manner.

In the case of either of the single chain specific binding domains,these domains may be used to screen for complementary domains capable offorming a two-domain specific binding member which has in vivoproperties as good as or equal to the SC104 antibody disclosed herein.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047 in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain specific binding memberis selected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Markset al. ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof. For example, specificbinding members based on the VL region shown in FIG. 1 c may be attachedat their C-terminal end to antibody light chain constant domainsincluding human Cκ or Cλ chains. Similarly, specific binding membersbased on VH region shown in FIG. 1 a or 1 b may be attached at theirC-terminal end to all or part of an immunoglobulin heavy chain derivedfrom any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of theisotype sub-classes, particularly IgG1 and IgG4.

SC104 has been demonstrated to cause the death of tumour cell-lines insuspension, and to cause the specific onset of apoptosis or programmedcell-death in colorectal tumours and cells derived from disaggregatedtumour tissue. Thus, specific binding members of the present inventioncan be used as therapeutics to inhibit the growth of, or induceapoptosis in, tumours. Apoptosis is the process by which a cell activelycommits suicide. It is now well recognised that apoptosis is essentialin many aspects of normal development and is required for maintainingtissue homeostasis. However, cell death by suicide, sometimes referredto as programmed cell death, is needed to destroy cells that represent athreat to the integrity of the organism. There are two differentmechanisms by which a cell commits suicide by apoptosis. One istriggered by signals arising from within the cell, the other by externalsignals (e.g. molecules) which bind to receptors at the cell surface.

Specific binding members of the present invention can be used in methodsof diagnosis and treatment of tumours in human or animal subjects.

When used in diagnosis, specific binding members of the invention may belabelled with a detectable label, for example a radiolabel such as ¹³¹Ior ⁹⁹Tc, which may be attached to specific binding members of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include enzyme labels such as horseradishperoxidase. Labels further include chemical moieties such as biotinwhich may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin.

Although specific binding members of the invention have in themselvesbeen shown to be effective in killing cancer cells, they mayadditionally be labelled with a functional label. Functional labelsinclude substances which are designed to be targeted to the site ofcancer to cause destruction thereof. Such functional labels includetoxins such as ricin and enzymes such as bacterial carboxypeptidase ornitroreductase, which are capable of converting prodrugs into activedrugs. In addition, the specific binding members may be attached orotherwise associated with chemotherapeutic or cytotoxic agents, such ascalicheamicin, or radiolabels, such as ⁹⁰Y or ¹³¹I.

Furthermore, the specific binding members of the present invention maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially, dependent upon the condition to betreated. Thus, the present invention further provides productscontaining a specific binding member of the present invention and anactive agent as a combined preparation for simultaneous, separate orsequential use in the treatment of a tumour. Active agents may includechemotherapeutic or cytotoxic agents including, 5-Fluorouracil,cisplatin, Mitomycin C, oxaliplatin and tamoxifen, which may operatesynergistically with the binding members of the present invention. Otheractive agents may include suitable doses of pain relief drugs such asnon-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol,ibuprofen or ketoprofen) or opitates such as morphine, or anti-emetics.

Whilst not wishing to be bound by theory, the ability of the bindingmembers of the invention to synergise with an active agent to enhancetumour killing may not be due to immune effector mechanisms but rathermay be a direct consequence of the binding member binding to cellsurface bound sialyltetraosyl carbohydrate.

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember. The pharmaceutical composition may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, diluent,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

It is envisaged that injections will be the primary route fortherapeutic administration of the compositions although delivery througha catheter or other surgical tubing is also used. Some suitable routesof administration include intravenous, subcutaneous and intramuscularadministration. Liquid formulations may be utilised after reconstitutionfrom powder formulations.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded. Where the formulation is a liquid it may be, for example, aphysiologic salt solution containing non-phosphate buffer at pH 6.8-7.6,or a lyophilised powder.

The composition may also be administered via microspheres, liposomes,other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semi-permeable polymermatrices in the form of shared articles, e.g. suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al,Biopolymers 22(1): 547-556, 1985), poly(2-hydroxyethyl-methacrylate) orethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982). Liposomescontaining the polypeptides are prepared by well-known methods: DE3,218, 121A; Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al,PNAS USA, 77: 4030-4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046;EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045 and4,544,545. Ordinarily, the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal rate of the polypeptide leakage.

The composition may be administered in a localised manner to a tumoursite or other desired site or may be delivered in a manner in which ittargets tumour or other cells.

The compositions are preferably administered to an individual in a“therapeutically effective amount”, this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.The compositions of the invention are particularly relevant to thetreatment of existing tumours, especially cancer, and in the preventionof the recurrence of such conditions after initial treatment or surgery.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16^(th) edition, Oslo, A. (ed),1980.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration. In general, a serum concentration ofpolypeptides and antibodies that permits saturation of receptors isdesirable. A concentration in excess of approximately 0.1 nM is normallysufficient. For example, a dose of 100 mg/m of antibody provides a serumconcentration of approximately 20 nM for approximately eight days.

As a rough guideline, doses of antibodies may be given weekly in amountsof 10-300 mg/m². Equivalent doses of antibody fragments should be usedat more frequent intervals in order to maintain a serum level in excessof the concentration that permits saturation of the sialyltetraosylcarbohydrate.

The dose of the composition will be dependent upon the properties of thebinding member, e.g. its binding activity and in vivo plasma half-life,the concentration of the polypeptide in the formulation, theadministration route, the site and rate of dosage, the clinicaltolerance of the patient involved, the pathological condition afflictingthe patient and the like, as is well within the skill of the physician.For example, doses of 300 μg of antibody per patient per administrationare preferred, although dosages may range from about 10 μg to 6 mg perdose. Different dosages are utilised during a series of sequentialinoculations; the practitioner may administer an initial inoculation andthen boost with relatively smaller doses of antibody.

This invention is also directed to optimise immunisation schedules forenhancing a protective immune response against cancer.

The binding members of the present invention may be generated wholly orpartly by chemical synthesis. The binding members can be readilyprepared according to well-established, standard liquid or, preferably,solid-phase peptide synthesis methods, general descriptions of which arebroadly available (see, for example, in J. M. Stewart and J. D. Young,Solid Phase Peptide Synthesis, 2^(nd) edition, Pierce Chemical Company,Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice ofPeptide Synthesis, Springer Verlag, New York (1984); and AppliedBiosystems 430A Users Manual, ABI Inc., Foster City, Calif.), or theymay be prepared in solution, by the liquid phase method or by anycombination of solid-phase, liquid phase and solution chemistry, e.g. byfirst completing the respective peptide portion and then, if desired andappropriate, after removal of any protecting groups being present, byintroduction of the residue X by reaction of the respective carbonic orsulfonic acid or a reactive derivative thereof.

Another convenient way of producing a binding member according to thepresent invention is to express the nucleic acid encoding it, by use ofnucleic acid in an expression system.

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acidincludes DNA and RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a specific binding member of theinvention as defined above. Examples of such nucleic acid are shown inFIGS. 1 a, 1 b and 1 c. The skilled person will be able to determinesubstitutions, deletions and/or additions to such nucleic acids whichwill still provide a specific binding member of the present invention.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone nucleic acid as described above. The present invention also providesa recombinant host cell which comprises one or more constructs as above.As mentioned, a nucleic acid encoding a specific binding member of theinvention forms an aspect of the present invention, as does a method ofproduction of the specific binding member which method comprisesexpression from encoding nucleic acid therefor. Expression mayconveniently be achieved by culturing under appropriate conditionsrecombinant host cells containing the nucleic acid. Following productionby expression, a specific binding member may be isolated and/or purifiedusing any suitable technique, then used as appropriate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli. The expression of antibodies and antibodyfragments in prokaryotic cells such as E. coli is well established inthe art. For a review, see for example Plüickthun, Bio/Technology9:545-551 (1991). Expression in eukaryotic cells in culture is alsoavailable to those skilled in the art as an option for production of aspecific binding member, see for recent review, for example Reff, Curr.Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech.6:553-560 (1995).

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2^(nd)Edition, Cold Spring Harbor Laboratory Press (1989). Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Ausubel et al. eds., ShortProtocols in Molecular Biology, 2^(nd) Edition, John Wiley & Sons(1992).

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. The introductionmay be followed by causing or allowing expression from the nucleic acid,e.g. by culturing host cells under conditions for expression of thegene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

EXAMPLES

The invention will now be described further in the followingnon-limiting examples. Reference is made to the accompanying drawings inwhich:

FIG. 1 a shows the nucleic acid and amino acid sequences of the SC104antibody heavy chain variable domain and a part of the constant region,FIG. 1 b shows the identical sequence to that outlined in FIG. 1 a butthe Kabat numbering system has been employed, and FIG. 1 c shows thenucleic acid and amino acid sequences of the SC104 antibody light chainvariable domain and a part of the constant region. FIG. 1 d demonstratesthat the chimeric version of the SC104 antibody, produced by atransfected cell line, binds the target cell line.

FIG. 2 a is a graph demonstrating binding of SC104 to a panel of celllines (Colo205, C170, MCF-7, MDA-231, MDA-435, T47D, ZR75, MKN45, RID9,T24, A431, PA1 and OAW28 (obtained from ECACC)). Cells were stained byELISA and results are expressed as absorbance (405 nm) for each cellline. FIG. 2 b is a graph demonstrating binding of SC104 to thefollowing cell lines: C170, HT-29, LoVo, Colo205, MKN45, RID9 and A431.Cells were stained by indirect immunofluorescence and analysed by flowcytometry. Results are expressed as X-mean for each cell line. SC101/29is supplied by Scancell as a positive control and recognisesLewis^(y/b). An isotyped matched antibody is used as a negative control.

FIG. 3 is a graph demonstrating binding of monoclonal antibodies tofreshly disaggregated colorectal tumour cells, as assayed by indirectimmunofluorescence and analysed by flow cytometry. Each point refers tothe mean fluorescence for an individual tumour.

FIG. 4 is a graph demonstrating binding of SC104 mab to purified CEA,C14 antigen and tumour glycolipid extract. The C14 antigen (90 KDaglycoprotein purified from saliva by affinity chromatography on C14monoclonal), CEA (180 KDa glycoprotein purified from colorectal tumourlive metastases by affinity chromatography with 365 mab) and glycolipidextract (glycolipid extracted in 3:1 methanol chloroform w/v fromcolorectal tumours) were dried onto microtitre plates by overnightincubation at 37° C. Binding of SC104 mab was assayed by ELISA andresults expressed as absorbance 405 nm. SC101 and anti-CEA antibodiesare included as positive controls.

FIG. 5 shows HPTLC plates showing A. SC104 immunostaining of fractionscollected from 36 ml bed volume column. Lane W is the whole C170 extractprior to fractionation. The first 6×10 ml fractions collected arespotted in lanes 1 to 6 while the last 14 lanes represent the next 14×3ml fractions collected. The SC104 antigen can be located in fraction 6,with R_(F)0.53. B. Secondary antibody only shows no band indicating thatbinding is specific to SC104.

FIG. 6 is a HPTLC plate showing (A) orcinol staining of fraction 6revealing three bands with RF values between R_(F)0.50 and 0.61. (B)SC104 immunostaining of the same fraction gave three tight bands betweenR_(F)0.36 and 0.39.

FIG. 7 is a HPTLC plate showing SC104 immunostaining of whole C170extract (lane W), fraction 6 (lane 6) and de-glycosylated lipid (laneL). Antigen was observed in the whole extract and fraction 6 (R_(F)0.38to 0.46), but no longer detected in the lipid fraction followingde-glycosylation by ceramide glycanase.

FIG. 8 is a HPTLC plate showing SC104 immunostaining of partiallypurified antigen. Pencil marks indicate the location of lipid detectedby iodine vapour staining. Lanes 1 and 2 contain the ceramide glycanasetreated antigen following separation into the aqueous and organic phasesrespectively. Released oligosaccharides partition into the aqueous phaseand free lipids into the organic phase. Any undigested glycolipids willpartition into either phase depending on overall polarity. Lanes 3 and 4are the same aqueous and organic partitioned fractions in the absence ofceramide glyvcanase. Antigen is detected (R_(F)0.48 and 0.40) prior tode-glycosylation but not following oligosaccharide removal.

FIG. 9 is a HPTLC plate showing SC104 immunostaining (A) and orcinolstaining of (B) of simple (lane S), glycolipid (lane G) and phospholipid(lane P) fractions from C170 cells. Antigen is detected in the simpleand phospholipid fractions (R_(F)0.54, 0.51 and 0.41) but not theneutral glycolipid portion. Orcinol staining reveals two distinct bandsin the phospholipid fraction (R_(F)0.54 and 0.51) that co-migrate withthe antigen.

FIG. 10 shows 2-dimensional HPTLC plates of C170 whole lipid extractfollowed by orcinol (A) or SC104 immunostaining (B). The first dimensionwas developed in 50:40:10 chloroform:methanol:CaCl₂ (0.5% w/v) and thesecond dimension in 10:4:2:2:1 chloroform:acetone:methanol:aceticacid:water. The antigen migrates with RF0.48 in the 1^(st) dimension anddoes not migrate in the 2^(nd). An orcinol stained band is again seen toco-migrate with the antigen.

FIG. 11 is a HPTLC plate showing SC104 immunostaining of antigen with(lanes 1 and 2) and without (lane 3) de-sialylation by neuraminidasedigestion. De-sialylation resulted in more intense staining of the lesspolar (R_(F)0.46) cluster and a decrease in staining of the more polar(R_(F)0.62) cluster.

FIG. 12 shows a HPTLC plate of C170 lipid fractions from silica column.Plates were probed with SC104 (A), secondary antibody only (B) orstained with orcinol (C) or ninhydrin (D). Antigen was detected in thewhole extract (lane W), and the sialylated glycolipid fractions (lanesN¹, N² and N³). No antigen was observed in either the simple (lane S) orphospholipid/neutral glycolipid fraction (lane P).

FIG. 13 is a graph demonstrating that whilst the SC104 antigen was seento cross-react with SC104 it did not cross-react with an anti-sialylLewis^(a) (a biomolecule previously evaluated for clinical potential) or19/9 antibody.

FIG. 14 demonstrates, using an SC104-Protein A sepharose column, a bandfor the SC104 immuno-purified antigen at between 50-75 KDa as identifiedon a Silver stained gel.

FIG. 15 shows the purified SC104 fraction from sputum competing withSC104 biotin for binding to C170 cells.

FIG. 16 is a histogram demonstrating the effect of SC104, or control791T/36 antibody, on C170 tumour cells. Cells were stained with FITClabelled Annexin and propidium iodide and then analysed by dual colourflow cytometry. Results are expressed as the % of cells staining withannexin, PI or both. SC101/29 is included as a positive control.

FIG. 17 is a graph to show FITC-z-FMK-vad activation of pan caspaseafter 6 hr exposure to adherent C170 cells. The results are expressed asX-mean.

FIG. 18 demonstrates caspase 6 activation on adherent cells treated withthe SC104 antibody. SC101 antibody treated cells are included as anegative control.

FIG. 19 demonstrates that if SC104 treated cells (A) are also exposed to3 uM z-FMK-vad inhibitor (B) cell death can be significantly reduced asassayed using annexin V FITC and propidium iodide.

FIG. 20 shows graphs showing the % viability (number of cells exposed tothe drug/number of cells exposed to control) if of a range of tumourcell lines (Colo205, C170, HT29 and LoVo). The number of viable cellswas determined by MTS and optical density reading at 490 nm.

FIG. 21 demonstrates IC₅₀ fits for the tumour cell lines, C170, Colo205,HT-29 and LoVo extrapolated from typical cell viability results (%viability=number of cells exposed to the drug/number of cells exposed tocontrol) as determined by MTS and optical density reading at 490 nm.

FIG. 22 is a graph to show that the fixation of tumour cells, either byCellfix or glutaraldehyde does not significantly alter the binding ofthe antibody to the tumour cell line C170 as compared to those untreatedin media alone. Cells were stained by indirect immunofluorescence,analysed by flow cytometry and the results expressed as mean linearfluorescent values.

FIG. 23 a shows that SC104 does not bind in a homophilic nature in theabsence of antigen. Microtitre plates were coated with goat anti-mouseIgG Fc specific antibody prior to adding increasing concentrations ofSC104 antibody. Bound SC104 antibody was detected by ELISA with goatanti-mouse horse radish peroxidase and TMB. To determine if SC104 couldbind to itself SC104 biotin was added and its binding detected withSA-HRP/TMB. Controls included, SC 104 which could not be detected withSA-HRP/TMB but both SC104 and SC104 biotin could be detected with goatanti-mouse HRP. Results are expressed as absorbance at 570 nm. FIG. 23 bthen shows that SC104 does bind in a homophilic fashion in the presenceof C14 antigen. Plates were coated with C14 antigen and SC104 added toeach well to the antigen. This was confirmed with goat anti-mouseHRP/TMB. SC104 biotin was then added at increasing concentrations andits binding was detected with SA-HRP/TMB. Results are expressed asabsorbance at 650 nm.

FIG. 24 is a graph demonstrating the effect of 5-Fluorouracil and SC104antibody on cells. C170 cells were exposed to SC104 or control 791T/36antibody (not shown) and 5-FU. The number of cells was determined by MTSand optical density reading at 490 nm.

FIG. 25 is a graph demonstrating the effects of 5-FU, Cisplatin,Mitomycin C, Oxaliplatin and Tamoxifen on C170 cells either alone or incombination with SC104 antibody, or with SC104 antibody alone.

FIG. 26 shows graphs demonstrating the effect of SC104, 5-FU/leucovorinand a combination of SC104 and 5FU/leucovorin on the growth of C170xenografts growing in nude mice. a) Growth of C170 xenografts wasmeasured at days 7, 9, 12, 14 and 16 by measurement of cross-sectionalarea (mm²) when animals were treated with either SC104 ip (0.2 mg),control antibody ip (0.2 mg) and 5-FU/leucovorin (12.5 mg/Kgiv) or SC104ip (0.2 mg) and 5-FU/leucovorin (12.5 mg/Kg iv) on days 1, 3, 5, 7, 21,22. b) A survival plot demonstrating the effect of SC104,5-FU/leucovorin or the combination of SC 104 and 5-FU on the survival ofnude mice expressing C170 xenografts. Animals were treated with eitherSC 104 ip (0.2 mg), control antibody ip (0.2 mg) and 5-FU/leucovorin(12.5 mg/Kgiv) or SC104 ip (0.2 mg) and 5FU/leucovorin (12.5 mg/Kgiv) ondays 1, 3, 5, 7, 21, 22. c) Animals were weighed on days 7, 14, 21, 28and 36 following treatment with SC104 ip (0.2 mg), control antibody ip(0.2 mg) and 5-FU/leucovorin (12.5 mg/Kgiv) or SC104 ip (0.2 mg) and5-FU/leucovorin (12.5 mg/Kg iv) on days 1, 3, 5, 7, 21, 22.

FIG. 27 is a graph demonstrating the effect of SC104, 5 FU/leucovorinand a combination of SC104 and 5FU/leucovorin on the growth of C170xenografts growing in nude mice. Mice were treated with % FU/leucovorin25 mg/Kg iv on days 1, 3, 5, 7, 21, 22 and with SC104 antibody (0.2 mg)three times per week staring on day 5. Growth of C170 xenografts wasmeasured at days 7, 9, 12, 14 and 16 by measurement of cross-sectionalarea (mm²) when animals were treated with either SC104 ip (0.2 mg),control antibody ip (0.2 mg) and 5FU/leucovorin or SC104 ip (0.2 mg) and5FU/leucovorin. A graph demonstrating the effect of SC101,5FU/leucovorin or the combination of SC101 and 5FU on the survival ofnude mice expressing C170 xenografts.

Example 1 Production of SC104 Monoclonal Antibody Methods

Immunisation: A range of isolated tumour cell lines were used in theimmunisation protocol for the production of SC104 following the regimeoutlined in the Table 1. BALB/c female mice (6-12 weeks old, Bantin andKingman, Hull) were immunised with 100% suspension of C146, C168, C170and JW cells at 0, 4, 13 and 14 months BALB/c mice respectively. A celldensity of 5×10⁶ cells/ml was used for the first two immunizations,while the density was reduced to 5×10⁵ cells/ml for the second twoimmunizations. The cells were suspended in Freund's complete adjuvant inthe first instance with the second and subsequent immunizations usingFreund's incomplete adjuvant. 5 days after the final immunisation thespleen cells were harvested and fused with PSNS1 cells.

TABLE 1 Immunisation protocol Day Cell Type Cell Number Route ofadministration 0 C170 5 × 10⁶ IP 42 C146 5 × 10⁶ IP 56 Colo205 5 × 10⁵IP 96 JW 5 × 10⁵ IP

Hybridoma Production

The mouse was sacrificed by cervical dislocation and the spleen removedaseptically. The spleen was transferred to a petri dish containing serumfree RPMI (20 ml, 37° C.) and the cells gently released into the medium.The tissue debris was allowed to settle from the cell suspension undergravity for two minutes. The individual splenocyte cells remaining insuspension were then recovered by centrifugation, resuspended in serumfree RPMI and counted. The harvested P3NS1 cells were counted andresuspended. The two cell types were mixed in a ratio of 1:10 cells,P3NS1: splenocytes. The cell mixture was recovered as a pellet andloosened by gentle tapping. Warm polyethlyene glycol 1500 was added (50%solution commercially available from Sigma chemicals) over 1 minute,followed by incubation at room temperature (1 min). Warm serum freemedia was then added over a further minute followed by the slow additionof a further 20 ml of serum free medium. The resuspended cells wererecovered as a pellet and gently dispersed in warm RPMI1640 containing15% foetal bovine serum and HAT selection agents. The cell suspensionwas aliquoted over 96-well plates previously coated with rat peritonealexudate cells (PECS). The plates were then incubated at 37° C., 5% CO₂,95% air until colonies of surviving hybridoma cells could be observed.The production of colon tumour specific antibody was screened for usingC170 cells as the primary layer in a non-competitive sandwich ELISA.Cells from a number of positive wells were pooled and plated out across96-well plates at cell densities of 5, 2.5, 1 and 0.5 cells/well (100μl/well). The plates were incubated at 37° C., 5% CO₂, 95% air until themedia in wells with colonies turned orange.

Screening Procedures

ELISA. Antibody responses in the serum of immunised mice were assayed bytitration using a standard non-competitive ELISA procedure. 96-welltissue culture plates were coated with C170 cells at a cell density of5×10⁵ cells/ml (100 μl/well) in 10% foetal bovine serum in RPMI 1640.The plates were incubated overnight at 37° C., 5% CO₂, 95% air. Thecells were then washed twice in phosphate buffered saline (pH7.3, PBS)prior to being fixed with 0.5% glutaraldehyde in PBS (10 min, 25° C.,100 μl/well). Remaining non-specific binding sites were blocked byincubation for 1 hr with 1% bovine serum albumin (fraction V from SigmaChemicals Ltd.) in PBS. The cells were washed three times with a washingsolution consisting of 0.05% Tween 20 in phosphate buffered salinebefore assaying post-boost serum for binding to C170 cells using anon-competitive sandwich ELISA. The pre-immunisation serum was used as anegative control.

Immunohistochemistry. Binding of hybridoma supernatants to tumourtissues was determined by indirect immunoperoxidase staining of frozencolorectal tumour sections. Tissue sections (5 μm) of cryopreservedtumour were treated with 0.3% H₂O₂ in 0.1% NaN₃ for 15 min to inhibitendogenous peroxidase. This was followed by incubation at roomtemperature with 10% human serum and 1% BSA prepared in PBS, for 30 min,and then the hybridoma supernatants were added at saturating levelswhich gave minimal non specific background staining for a further 30min. The bound antibody was detected with rabbit anti-mouse Igconjugated to peroxidase (Dako Ltd., Bucks., UK) and following extensivewashing the slides were stained with 0.05% diaminobenzidine and 0.01%H₂O₂ in 0.05M Tris-HCl, pH7.6 and counterstained with haematoxylin.

Results

SC104 is a monoclonal antibody (mab) that was raised by immunisation ofmice with 4 colorectal cancer cell lines. It was screened byimmunohistochemistry against colorectal tumour sections and by ELISAagainst one of the immunizing cell lines. It was cloned three times andwas shown to be a mouse IgG1 mab. Table 2 shows that it binds to C170cells with a higher intensity than a positive control antibodyrecognising Lewis^(y/b) hapten. It also showed intense staining ofcolorectal tumours in comparison to either the Lewis^(y/b) antibody oran anti-CEA antibody. It showed similar weak staining of normal colontissues to an anti-CEA antibody.

TABLE 2 Screening of SC104 mab C170 ELISA Immunohistochemistry^(a)Antibody (OD 405 nm) Colorectal Tumour Normal colon SC104 0.301 3+ +Anti-Lewis ^(y/b) 0.161 2+ Mucin staining only Anti-CEA 0.001 2+ + Noantibody 0.001 − − ^(a)− negative, + minimal, 2+ strong, 3+ very strong

Example 2 Sequence of the SC104 Antibody Methods

Amplification and sequencing of the heavy and light kappa chain variableregions of the mouse immunoglobulin SC104. Total RNA was isolated fromthe hybridoma SC104/1E9 after checking previously for antibodyproduction by ELISA. cDNA synthesised from 5 μg of the total RNA wasused as a template for the amplification of the heavy and light chainvariable domains of SC104. The following forward oligonucleotides thatanneal to the leader sequence and reverse oligonucleotides to the CH1domain of the constant region of each chain were utilised respectivelyin the PCR reactions with the high fidelity enzyme pfu turbo(Stratgene).

Forward Primers 5′-ATG AGA GTG CTG ATT CTT TTG TG-3′ Heavy chain 5′-ATGGAT TT(A/T) CA(A/G) GTG CAG ATT (A/T)TC AGC TTC-3′ Kappa chain ReversePrimers 5′-CCC AAG CTT CCA GGG (A/G)CC A(A/G)(G/T) GGA TA(A/G) ACGG(A/G)T GG-3′ Heavy chain 5′-CCC AAG CTT ACT GGA TGG TGG GAA GAT GGA-3′Kappa chain

The amplified fragments were cloned into the TA vector pCR2.1(Invitrogen). Clones containing insert were identified by restrictionanalysis and confirmed by DNA sequencing with the primers T7 and M13reverse (Lark Technologies). Sequences were analysed and the followingcomplimentary determining regions (CDR'S) identified for the heavy andlight chain of the antibody SC104 (FIGS. 1 a, b and c).

To verify that analysis of sequencing and translation was correct forboth chains 10 μg of purified and concentrated SC104 antibody wasdenatured, separated on a 0.75 mm thick 8% SDS-PAGE gel byelectrophoresis and transferred onto PVDF by semi dry electroblotting.After staining with Amido black the Kappa (25 KDa) and Heavy (50 KDa)chains were isolated and N terminally sequenced by Edman degradation(Alta Biosciences). Protein sequencing confirmed the analysed DNA andtranslated sequences for both chains.

Heavy Chain Variable region (55 bp-414 bp) CDR1 (130 bp-162 bp) G  Y  S  I  T  S  G  Y  S  W  H GGCTACTCCATCACGAGTGGTTATAGTTGGCACAccording to Kabat numbering (145 bp-162 bp) S  G  Y  S  W  HAGTGGTTATAGTTGGCAC CDR2 (205 bp-252 bp) H  I  H  F  S  G  R  P  T  Y  N  P  S  L  S  SCACATTCACTTCAGTGGTAGACCTACTTACAATCCATCTCTCAGCAGT CDR3 (349 bp-381 bp)K  G  K  G  S  D  D  G  L  N  Y AAGGGAAAAGGTTCCGACGATGGTTTGAACTAC

Signal leader sequence (1 bp-54 bp) FR1 (55 bp-129 bp) FR2 (163 bp-204bp) FR3 (253 bp-348 bp) FR4 (382 bp-414 bp) CH1 (415 bp onwards)

Kappa Light Chain Variable region (67 bp-384 bp) CDR1 (136 bp-165 bp)S  A  S  S  S  L  S  Y  I  H AGTGCCAGCTCAAGTTTAAGTTACATACAC CDR2 (211bp-231 bp) D  T  S  N  L  A  S GACACATCCAACCTGGCTTCT CDR3 (328 bp-354bp) F  Q  G  S  E  Y  P  L  T TTTCAGGGGAGTGAGTATCCACTCACG

Signal Leader sequence (1 bp-66 bp) FR1 (67 bp-135 bp) FR2 (166 bp-210bp) FR3 (232 bp-327 bp) FR4 (355 bp-384 bp) CH1 (385 bp onwards)

Example 3 Expression of Chimeric SC104 Monoclonal Antibody

To verify expression of functional antibody from the DNA sequences theAfe1 and BsiW1 restriction sites were incorporated into the heavy andlight chain variable regions at the end of framework 4 within pCR2.1 bysite directed mutagenesis using the designed complimentaryoligonucleotides.

Heavy chain variable Afe1 Forward Primer 5′-C TCA GTC ACC GTC TCTAGC GCT AAA ACG ACA CCC CCA CC-3′ Reverse Primer 5′-GG TGG GGG TGT CGTTTT AGC GCT AGA GAC GGT GAC TGA G-3′ Light chain variable BsiW1 ForwardPrimer 5′-CC AAG CTG GAA ATG ACA CGT ACG GAT GCT GCA CCA ACT G-3′Reverse Primer 5′-C AGT TGG TGC AGC ATC CGT ACG TGT CAT TTC CAG CTTGG-3′

The murine heavy and light chain variable regions of SC104 were excisedfrom pCR2.1 and cloned into the HindIII/Afe1 and BanHI/BsiW1 sites ofthe mammalian expression vector pDCORIG IB fused inframe with the humanFc constant region of each chain respectively. This plasmid wasidentified by restriction analysis and confirmed by DNA sequencing.

CHO—S was stably transfected with 15 μg of the above plasmid andgenejuice (Novagen) according to the manufacturer's recommendations.Transfectants were selected in medium containing Zeocin (300 μg/ml) for14 days. Expression of functional chimeric SC104 antibody secreted fromthe transfectants was confirmed by binding to the cell surface of thecell line C170 and flow cytometry (FIG. 1 d).

In brief after washing in RPMI/10% FCS C170 cells were incubated for 30minutes at 4° C. with either spent medium containing the chimericantibody or medium alone. Cells were washed and incubated for a further30 minutes at 4° C. with a FITC conjugated rabbit anti-human IgGantibody specific for the CH2 domain (DAKO, F0123). Cells were washedagain, resuspended and analysed by flow cytometry.

Example 4 Binding Studies Using SC104 Monoclonal Antibody Experiment 1Methods

Immunohistochemistry staining of normal and tumour tissues. Humantissues were obtained from an Inveresk approved supplier. Each tissueused was snap frozen in liquid nitrogen and stored at approximately −70°C. (±10° C.). Cryostat sections were cut and placed onto super frostplus slides. All tissue samples were treated with an antigen markerappropriate for each tissue in order to confirm the preservation ofantigens in that tissue. The markers used were keratin for epithelialbearing tissues, CD45 for all lymphoid tissues, and desmin for allcardiac and skeletal muscle. Each tissue was examined from threeunrelated donors. The method of staining employed was an indirect, 2 or3-stage method, using secondary and tertiary antibodies together with anavidin-biotin-peroxidase complex. Endogenous peroxidase activity wasblocked using hydrogen peroxide. Endogenous biotin was blocked bytreating all tissue sections with a sequence of avidin-biotin. When atertiary antibody was used, all tissue sections were treated with normalswine serum, which inhibits the binding of tertiary antibodies to thetissues. Validation of the immunohistochemical staining method wasdetermined by staining on positive control tissues, the absence onnegative controls and the effect of fixation on staining of controltissues. SC104 was tested against 6 donors of colon tumour and one ofheart at concentrations of 0 or 50 μg/ml. Tissues were fixed in eitheracetone or neutral buffered formalin. Two samples of colon tumourstained well enough for use as a positive control and there was nostaining in heart. Neutral buffered formalin was found to be the optimumfixative. The lowest concentration of SC104 giving the maximum stainingintensity was 1 μg/ml and this concentration was used for subsequentwork.

Results

SC104 was screened for binding to a range of frozen tumour and normaltissue sections (Table 3). There was positive staining of the neoplasticepithelium of the colon, endometrial, oesophageal, parotid salivarygland and stomach and less intense staining of small numbers ofepithelial cells in three of the six breast tumours. Positive stainingwas recorded in the epithelium of normal large intestine, parotidsalivary gland, tonsil and uterine cervix. Less intense staining wasrecorded in small numbers of transitional epithelial cells in theurinary bladder, scattered thymic lymphocytes of a single donor,glandular epithelial cells of the skin, epithelial cells of theprostate, breast and fallopian tube, ovarian follicular cells, alveolarlining cells of the lung and small numbers of glial cells from the brainof one donor. Mucus stained positively in the stomach and smallintestine. No specific staining was recorded in normal human heart,kidney, placenta or spleen. These results suggest that the antigen canbe expressed by a range of epithelial cells but that it is predominantlylocalised within the gastrointestinal tract. The immunohistochemistrysuggested that the antigen was expressed more strongly on colorectaltumours than adjacent normal tissues.

TABLE 3 Immunoreactivity of SC104 antibody with frozen tumour and normaltissue sections Anti-EGF SC104 Tissue Mab binding Comments on stainingTumours Lung 6/6 0 Oesophageal 4/4 4/4 Breast 0 3/6 Renal ND 0 Colon 3/88/8 Ovary 0 0 Parotid 0 2/2 Prostate 0 0 Stomach 0 4/4 Testes ND 0/1Endometrial 1/2 2/2 Normal Tissues Brain 0 1/2 Small number of scatteredglial cells from the brain of one donor Breast 3/3 2/3 Epithelial cellsscattered moderate Fallopian tube 0 2/3 Epithelial cells scatteredmoderate Heart 0 0 Kidney 0 0 Large intestine 3/3 3/3 Epithelial cells,mucous, diffuse mild to moderate Lung 0 2/3 Alveolar lining cells onedonor minimal and one donor mild Ovary 0 3/3 Follicular cells scatteredand mild Placenta 3/3 0 Prostate 3/3 3/3 Epithelial cells scatted andmoderate Parotid salivary gland 3/3 3/3 Moderate Skin 3/3 2/3 Diffuseminimal staining Small intestine 0 3/3 Mucus superficial, diffuseminimal Spleen 0 0 Stomach 0 2/3 Mucus superficial, diffuse mild Thymus0 3/3 Epithelial cells, Hassall's corpuscle, membrane scattered mildTonsil 0 3/3 Keratinised epithelium, membrane, scattered mild tomoderate. Urinary Bladder 2/3 2/2 Epithelial cells transitionalepithelium, membrane scattered mild Uterine cervix 3/3 3/3 Epithelialcells, membrane, diffuse mild to moderate Notes: ND not done.

Experiment 2 Methods

Binding to tumour cell lines by indirect immunofluorescence and flowcytometric analysis. C170, Colo205, MKN45, R1D9, and 791T cells (105)were resuspended in 50 μl of SC104 mab (0-20 μg/ml) and incubated on icefor 20 min. After washing the samples 3 times in RPMI/10% FCS cells wereincubated with FITC labelled rabbit anti-mouse ( 1/50: Dako Ltd, Bucks,UK) antibody and incubated on ice for 30 minutes prior to analysis on aFACScan (Becton Dickinson, Sunnyvale, Calif.). Results are expressed asmean linear fluorescence (MLF).

Binding to tumour cells by ELISA. 96-well tissue culture plates werecoated with cells at a cell density of 5×10⁵ cells/ml (100 μl/well) in10% foetal bovine serum in RPMI 1640. The plates were incubatedovernight at 37° C., 5% CO₂, 95% air. The cells were then washed twicein phosphate buffered saline (pH7.3, PBS) prior to being fixed with 0.5%glutaraldehyde in PBS (10 min, 25° C., 100 μl/well). Remainingnon-specific binding sites were blocked by incubation for 1 hr with 1%bovine serum albumin (fraction V from Sigma Chemicals Ltd.) in PBS. Thecells were washed three times with a washing solution consisting of0.05% Tween 20 in phosphate buffered saline. Cells were incubated withSC104 (1 μg/ml) for 1 hr at room temperature and then bound antibodydetected with goat anti-mouse horse radish peroxidase and ABTS. Resultsare expressed as absorbance at 405 nm.

Results

Further characterisation of the antigen expression was performed byELISA on a range of tumour cell lines (FIG. 2 a). SC104 antibodypredominantly bound to cell lines of the gastrointestinal origin. SC104bound to C170, Colo205 and R1D9 cells with a MLF of 1500 to 4,000.

Experiment 3 Methods

Primary tumour binding. Tumour specimens were obtained at the time ofcolorectal cancer resection. Specimens were finely minced anddisaggregated with 0.05% collagenase (Type IV, Boehringer Mannheim,Lewes, UK) for 20 min at 37° C. The tumour cell suspension was removedand washed 3 times in Hanks balanced salt solution (Gibco BRL, Paisley,UK). Fresh collagenase was added to the remaining tissue and it wasreincubated for a further 20 min. This procedure was repeated twicebefore combining cells from all dissociations and resuspending them inDulbecco's medium containing 20% fetal calf serum (Gibco). Cells wereincubated for 1 hr at 4° C. with SC104 mab (1 μg). Cells were washedtwice and incubated for a further hr with FITC-conjugated rabbitanti-mouse immunoglobulin (Dako Ltd., Bucks., UK). Normal mouseimmunoglobulin was used as a negative control and this fluorescence wassubtracted from the fluorescence obtained with SC10 4. The cells werewashed 3 times prior to analysis on a FACScan (Becton Dickinson,Sunnyvale, Calif.). Results are expressed as mean linear fluorescence(MLF). Disaggregation of solid tumours yields a mixed population ofcells including red blood cells, lymphocytes, stromal cells, macrophagesand endothelial cells. The percentage of epithelial cells is measured bystaining of cytokeratin mab Cam 5.2. was only 22±13% (range 10-60).However, following forward angle light scatter gating to selectivelyanalyse cells in the malignant cell size range 79±4% (range 69-86) ofthe cells analysed were epithelial. Furthermore, the variation betweentumours was considerably reduced. The percentage of leukocytes asmeasured by staining with the anti-CD45 mab F-10-89 in the totalnucleate population was 74±16 (range 40-90). This was considerablyreduced to 5.5±5% (range 1-20) following FACS IV gating for malignantsize. The percentage of stromal cells in the population of cellsanalysed in the malignant size range was 3.5±3% (range 1-13).

Results

SC104 was also shown to bind strongly to >80% of freshly disaggregatedcolorectal tumour cells with a mean antigen density of 4×10⁵ antigensper cell (range 1.5−10×10⁶ antigen per cell; FIG. 3).

Experiment 4 Method

Tumour and normal membrane extract binding. Extranuclear extractions ofcolorectal and normal colonic membranes were produced and binding ofSC104 was assayed by ELISA.

Results

To further quantify the differential staining obtained byimmunohistochemistry, extranuclear membrane preparations of primarycolorectal tumours and normal colon from the resection margin wereproduced. ELISA staining of these membranes with SC104 revealed weakstaining of the normal colon whereas moderate to strong staining of thetumour membranes were observed with the mean T:N ratio being 6:1 (Table4).

TABLE 4 Relative binding of SC104 to tumour and normal membrane extractsBinding of SC104 mab to extranuclear membranes Colorectal Patient TumourNormal colon T:N ratio 1 0.001 0.001 — 2 0.437 0.068 6.6:1   3 0.2210.217 1:1 4 0.278 0.042 7:1 5 0.196 0.066 3:1 6 0.281 0.035 8:1 7 0.1020.139 1:1

Experiment 5 Methods

Binding to purified antigen preparations. The C14 antigen (90 KDaglycoprotein purified from saliva by affinity chromatography on C14monoclonal), CEA (180 KDa glycoprotein purified from colorectal tumourlive metastases by affinity chromatography with 365 mab) and glycolipidextract (glycolipid extracted in 3:1 methanol chloroform w/v fromcolorectal tumours) were dried onto microtitre plates by overnightincubation at 37° C. Binding of SC104 mab was assayed by ELISA asdescribed above.

The Lewis^(y) hapten and the type I and type II H blood group antigen(Sigma, Poole, Dorset) were dried microtitre plates by overnightincubation at 37° C. Binding of SC104 mab was assayed by ELISA asdescribed above.

Results

To try and identify the nature of the antigen recognised by SC104antibody, it was used to stain three different antigen preparations(FIG. 4). The first was CEA as this is a well known and immunogenicantigen expressed by colorectal tumours. No significant staining of CEAwas observed although the anti-CEA antibody showed good reactivityconfirming the presence of functional antigen. The second preparationwas a Lewis^(y/b) expressing glycoprotein extracted from saliva. Thisantigen is a 90 KDa glycoprotein that carries a wide range ofcarbohydrate residues. Both the anti-Lewis y/b antibody and SC104antibody bound to this glycoprotein whereas the anti-CEA antibody failedto bind. A similar result was obtained when a methanol/chloroform tumourglycolipid extract was assayed. These results suggested that SC104 wasrecognising a carbohydrate residue expressed on both glycoproteins andglycolipids and that it may be recognising Lewis^(y/b). To verify ifSC104 was not binding to a blood group antigen it was screened forbinding to Lewis^(y), H type I and type II blood group haptens by ELISA(Table 5). The anti-H antibody bound strongly to all three haptens andthe Lewis^(y/b) antibody to the Lewis^(y) hapten but SC104 failed toreact with any of these haptens.

TABLE 5 SC104 binding to blood group antigens Binding of antibodies toblood group antigens as measured by ELISA (OD 405 nm) H blood group Hblood group Antibody Lewis ^(y) Type I Type II SC104 0.057 0.087 0.076Anti-H 2.058 2.174 1.331 Anti-Lewis ^(y/b) 0.662 0.129 .097

Example 5 Identification of Tumour Glycolipid Recognised by SC104Monoclonal Antibody Experiment 1 Optimisation of Lipid Extraction andImmunostaining Protocols Methods

Lipid extraction from C170 tumour cells. A pellet of C170 cells (1 mlpacked cell volume) was obtained by trypsinization of a static adherenttissue culture. The pellet was washed in PBS and the excess bufferremoved by aspiration following centrifugation, then stored at −80° C.The pellet was extracted with chloroform:methanol (3:1, v/v, 19 ml). Theresultant emulsion was centrifuged at 8,000 rpm in a 50 ml solventresistant (Tefzel) centrifuge tube for 15 min at 4° C. The supernatantwas dried down using a rotary evaporator at 30° C. and resuspended in asmall volume (˜1 ml) chloroform:methanol:0.5% CaCl₂(aq) (50:40:10).HPTLC analysis of lipid extracts The sample was multiply spotted onto aMerck HPTLC plate and developed in chloroform:methanol:0.5% CaCl₂(aq)(50:40:10) as standard. The plates were dried and then placed in aniodine vapour tank. Bands were marked in pencil and the iodine allowedto evaporate off the plate over night. The plates were thenimmunostained in order to localise the antigen.

Immunostaining of developed HPTLC plates. For SC104 immunostaining, theplates were immersed in polyisobutyl methylmethacrylate (0.1% w/vsolution) hexane:chloroform (9:1) for 15 seconds then allowed to dry.The plates were then blocked in 3% BSA in PBS for 1 hr at roomtemperature, followed by incubation in either test antibody solution (10μg/ml) or BSA solution, for 1 hr at room temperature. The plates werethen washed three times in PBS/Tween-20 (0.1%), before being incubatedin rabbit anti-mouse HRP conjugate from Dako ( 1/250 in PBS) for 1 hr atroom temperature. The plates were then washed three times in PBS/Tween−20 (0.1%) and once in 10 mM Tris, 100 mM NaCl pH 7.0, 0.1% Tween anddeveloped in Sigma-FAST BCIP/NBT reagent.

Chemical staining of developed HPTLC plates. Orcinol staining wasemployed to detect the presence of carbohydrate moieties. DevelopedHPTLC plates were allowed to dry before spraying with orcinol reagentuntil fully coated. The plates were dried in a stream of hot air andincubated at 100° C. for 15 minutes. Ninhydrin staining was used todetect molecules containing free amino groups. Developed HPTLC platewere first allowed to dry and then dipped in ninhydrin solution (0.25%ninhydrin w/v in acetone) until fully wetted. The plate was then allowedto develop at room temperature for several hours.

Results

The antigen was found to be successfully extracted in 3:1chloroform:methanol mix, with no change in the number of bands detectedcompared to the original 2:1 solvent extraction. However, increasing thesolvent to 4:1 chloroform:methanol appeared to reduce the number ofbands detected by HPTLC and consequently was not employed for extractionpurposes. Early immunostaining experiments also employed a 60 secondincubation of the HPTLC plate in 0.1% w/v polyisobutyl methacrylate inhexane; however, it was found that it was necessary to reduce this to 15seconds to give reproducible detection of the antigen. Finally it wasfound necessary to incorporate a phosphate free wash of the HPTLC platesprior to incubation with the phosphatase substrate to ensure maximumsensitivity.

SDS-PAGE and Western blotting experiments have previously shown thatSC104 antigen could be detected in the whole cell extract, cell lysateand the insoluble pellet produced during the preparation of cellmembrane. The antigen was however not detected in the cell membranepreparation itself. In addition, HPTLC separation and immunostaining ofthe insoluble pellet produced in the membrane preparation was also ableto detect the antigen. This suggests that the antigen is lipid or a veryhydrophobic protein.

Experiment 2. Analysis of Antigen Containing Fraction by Silica ColumnChromatography Methods

Silica column fractionation of lipid extract. A gravity column (150 mmi.d.) was packed with a silica slurry in chloroform:methanol:0.5%CaCl₂(aq) (50:40:10). The C170 extract, re-dissolved in 1 mlchloroform:methanol:0.5% CaCl₂(aq) (50:40:10) was loaded onto the columnand eluted using the same solvent conditions. Fractions were collectedand analysed by HPTLC. SC104 immunostaining was employed to locate theantigen and chemical staining methods were used to further characterisethe fraction.

Ceramide glycanase digestion of antigen. Ceramide glycanase digestionwas used to release oligosaccharides from glycolipid molecules. Antigensolution in 50 mM sodium acetate, pH 5.0, 0.1% w/v sodium cholate wasmixed with ceramide glycanase solution (1 μl for 50 μl antigen) andincubated at 37° C. for 3 hrs. The de-glycosylated lipid was separatedfrom the released oligosaccharides by mixing with 25μlchloroform:methanol (2:1) followed by brief centrifugation. The upperaqueous phase was separated from the lower organic phase and the twodried down separately.

Results

Small scale fractionation of C170 lipid extract (obtained from 0.5 mlpacked cell volume) was demonstrated on a 10 ml bed volume silica columnusing 50:40:10 chloroform:methanol:CaCl₂ (0.5% w/v in aqueous solution)as the mobile phase. However, in order to allow antigen characterisationit was necessary to scale this method up. This was shown to be possibleusing ˜10 ml packed cell volume to obtain the lipid extract andincreasing the column volume to 36 ml.

Whole lipid extract was loaded onto the scaled up column, fractionscollected and the antigen-containing fraction detected by immunostaining(FIG. 5). An antigen with R_(F) of 0.53 was detected. HPTLC and chemicalstaining was then used to further characterise this fraction. Orcinolstaining of the HPTLC plate was used to detect the carbohydrate moietyof glycolipids. This revealed three bands with R_(F) values between 0.50and 0.61 and seemed to correspond to the initial R_(F) value of theantigen. However, immunostaining repeated concurrently with orcinolstaining revealed an unusual shift in the R_(F) of the antigen, with 3bands detected between R_(F)0.36 and 0.39 (FIG. 6). This may suggestthat the bands detected by orcinol staining do not correspond to theantigen but represent glycolipid contaminants.

In order to ascertain if this antigen was a protein, the fraction wasanalysed by SDS-PAGE and Western blotting. Silver staining of the geldid reveal very low levels of protein contamination; however Westernblotting failed to specifically detect any antigen in the preparationsuggesting that the antigen itself is not a protein.

An aliquot of the antigen-containing fraction was digested with ceramideglycanase to remove the carbohydrates. The glycans were separated fromthe lipid portion using 2:1 chloroform:methanol; with the free glycanspredicted to partition with the methanol and the de-glycosylated lipidspartitioning with the chloroform. Immunostaining and chemical stainingtechniques were then performed on HPTLC plates of intact glycolipid andde-glycosylated lipid. Immunostaining revealed the presence of antigen(R_(F)0.38-0.46) with the intact glycolipid and not the de-glycosylatedlipid (FIG. 7). However, later experiments demonstrated that thepartitioning of undigested antigen is highly variable, with the antigensometimes being detected in either the aqueous, organic or both phases.Thus, the failure to detect antigen in a ceramide glycanase treatedorganic fraction is inconclusive. It may be due to partitioning of theantigen into the aqueous phase, rather than ceramide glycanase removalof the antigen.

In order to confirm the above experiment it was repeated with both theorganic and the aqueous phase being analysed following digestion.Analysis of the samples by HPTLC and iodine staining demonstrated a lossof SC104 binding in both fractions following the removal ofoligosaccharides (FIG. 8). This indicates that SC104 recognises anintact glycolipid and that this recognition is lost followingoligosaccharide removal.

Phospholipids such as phosphatidylethanolamine, phosphatidyl serine andthe related lyso groups have free amino groups that can be detected byninhydrin staining. HPTLC and ninhydrin staining of both the intactglycolipid and the ceramide glycanase treated fraction revealed thepresence of phospholipids with R_(F) values between 0.51 and 0.54.However, this may be a contaminant that co-migrates with the antigen,particularly as the literature suggests that co-elution of phospholipidsand glycolipids is a common problem when employing a single silicacolumn purification method. This indicates that such a method may beinsufficient to resolve the antigen from the whole extract withsufficient purity to facilitate characterisation.

Experiment 3.

Fractionation of whole lipid extract into lipid sub-groups andlocalisation of antigen

Methods

Lipid was extracted from 2 ml packed cell volume C170 cells as describedabove. Following evaporation the extract was resuspended in ˜1 mlchloroform. A 10 ml bed volume silica column (150 mm i.d.) was preparedin 100% chloroform. The sample was added to the column and the columnwashed under gravity in the following solvents: 10 column volumeschloroform, to elute simple lipids; 40 column volumes acetone, to eluteneutral glycolipids; 10 column volumes methanol to elute gangliosidesand phospholipids. The fractions were collected and dried down viarotary evaporation and stored at 4° C. prior to analysis.

Neuraminidase digestion of antigen. Antigen solution (511) was combinedwith 5 μl 10× buffer solution, provided with the neuraminidase, and 10μneuraminidase solution. The final volume was brought up to 50 μl, mixedand incubated at 37° C. for 1 hr.

Chemical de-sialylation by acid hydrolysis. Antigen solution in 0.05 Msulphuric acid was heated at 80° C. for 2 hrs. The sample was thenallowed to cool and stored at 4° C. prior to analysis.

Results

In order to remove glycolipids from phospholipids, a 10 ml silica columnwas equilibrated with chloroform. Whole C170 lipid extract was obtainedfrom 2 ml packed cell volume. This was applied to the column and thecolumn washed with chloroform, to elute the simple lipids; acetone, toelute glycolipids; and finally methanol to elute phospholipids.

HPTLC separation and immunostaining detected at least three antigenbands in the whole extract and phospholipid fraction (R_(F)0.54, 0.51and 0.41) along with a single band in the simple lipid fraction(R_(F)0.51; FIG. 9). However, no antigen was detected in the glycolipidfraction. Ninhydrin staining revealed the presence of phospholipid inthe whole extract and phospholipid fraction only. However, orcinolstaining demonstrated that glycolipids were present in both theglycolipid and phospholipid fractions. Significantly, two distinct bandswere observed in the phospholipid fraction (R_(F)0.54 and 0.51) thatappear to co-migrate with the antigen, again indicating that the antigenmay carry carbohydrate moieties.

Further reading indicated that although the above method can be used toseparate neutral glycolipids from phospholipids, sialylated glycolipidsco-elute with phospholipids. This, along with the co-migration of theantigen and orcinol positive bands provided some evidence that theantigen may be a sialylated glycolipid; while the absence of antigen inthe neutral glycolipid fraction suggests that the glycolipid must besialylated in order to be recognised by SC104.

Further evidence that the antigen is a sialylated glycolipid and not aphospholipid was obtained by separating whole lipid extract using 2DHPTLC and analysing the plates via immunostaining and chemical staining.In the first dimension the HPTLC plate was developed in the standard50:40:10 chloroform:methanol:CaCl₂ solvent mixture. The plate was thenallowed to dry, rotated 90° and developed inchloroform:acetone:methanol:acetic acid:water 10:4:2:2:1. Immunostainingrevealed that very little migration of the antigen occurred in thesecond dimension (FIG. 10). This again demonstrated that the antigen isnot a neutral glycolipid, since neutral glycolipids migrate in acetone.Orcinol staining of carbohydrates again showed a band that co-migratedwith the antigen in both dimensions, while ninhydrin staining of freeamino groups demonstrated a compound that co-migrated in the firstdimension but which was then separated from the antigen in the seconddimension.

Neuraminidase digestion was used to detect the effect of sialic acidremoval on antibody recognition. Immunostaining of HPTLC separatedphospholipid/ganglioside sample before and after neuraminidase treatmentprovided two clusters of bands; with a more polar cluster at R_(F)0.46and a less polar at R_(F)0.62 (FIG. 11). Although the same bands weredetected before and after neuraminidase treatment there was a shift inthe intensity of the staining profile. Neuraminidase treatment producedmore intense staining at the R_(F)0.46 cluster and a decrease inintensity at the R_(F)0.62 cluster when compared to the untreatedcontrol. This suggests that although sialic acid removal was onlypartial, since the conditions had not been optimised, removal of sialicacids from the more polar R_(F)0.62 cluster led to formation of a lesspolar cluster of molecules, which are still detected by the antibody. Asthe least polar bands detected after partial neuraminidase digestionco-migrated with the least polar bands in the untreatedganglioside/phospholipid fraction it is assumed that this cluster mustitself correspond with a sialylated form, probably monosialylated, whilethe more polar cluster corresponds to a disialylated form.

Silica column chromatography was then employed to fractionatedifferentially sialylated glycolipids. A sample of lipid extract wasloaded onto the column and fractions were eluted to obtain simple lipid(chloroform); asialylated glycolipids and phospholipids(chloroform:methanol:acetone:acetic acid:water 52:8:8:18:4, followed bychloroform:methanol 4:1); monosialylated glycolipids(chloroform:methanol 2:3); disialylated glycolipids(chloroform:methanol:water 65:25:4) and finally polysialylatedglycolipids (chloroform:methanol:water 60:35:8). The fractions were thendried down and analysed by HPTLC followed by immunostaining and chemicalstaining (FIG. 12).

Orcinol and ninhydrin staining showed phospholipids and glycolipids wereboth present in the sialylated glycolipid fractions. However the antigenagain appears to co-migrate with the only orcinol positive glycolipidsand not the ninhydrin positive phospholipids.

HPTLC and immunostaining of these samples revealed three clusters ofantigen in the whole extract (R_(F)˜0.49, ˜0.38 and ˜0.24), with themost polar of these showing only very faint staining. No antigen wasobserved in the simple lipid fraction or the phospholipid/neutralglycolipid fraction, but antigen was detected in the sialylatedfractions. This confirms that the antigen is a sialylated glycolipid andnot a phospholipid. Two of the clusters of bands were detected in themonosialylated fraction (R_(F)˜0.49 and R_(F)˜0.38). In the disialylatedfraction faint staining is visible for the most (R_(F)˜0.24) and least(R_(F)˜0.49) polar clusters, with the most intense staining detected forthe middle cluster (R_(F)˜0.38). In the polysialylated fraction twoclusters are visible (R_(F)˜0.38 and R_(F)˜0.24). This suggests that theleast polar cluster is monosialylated, the middle cluster representsdisialylated structures and the most polar cluster has three or possiblyfour sialic acids.

Acid hydrolysis was then used to chemically de-sialylate the antigen.This method is more aggressive than enzymatic procedures and is morelikely to result in complete removal of sialic acids. HPTLC and SC104immunostaining demonstrated that chemical removal of sialic acidsresulted in the complete loss of SC104 binding, providing furtherevidence that sialylation of the antigen is necessary for antibodybinding.

SC104 therefore appears to bind to a mono, di and even polysialylatedglycolipid. However it does not bind to an asialo form. This indicatesthat the antigen must be monosialylated however the presence of furthersialic acids does not impinge on the binding of SC104. The increasedintensity of staining for the disialylated form also suggests thatdespite only requiring the presence of one sialic acid, the antigen ismost commonly found bearing two sialic acids. However, it is currentlyunclear if this is dependent upon the age and culture conditions of theC170 cells prior to harvesting.

Experiment 4. Comparison of Antigen to Commercially Available LipidStandards Methods

The SC104 antigen has been compared to a number of commerciallyavailable lipid standards. Examples of each standard were spotted ontoHPTLC plates and developed in 50:40:10 chloroform:methanol:CaCl₂ (0.5%w/v). Plates were also spotted with partially purified SC104 antigen.Migration of each standard was detected by iodine vapour staining andthe location of each marked on the plate. Plates were then probed withSC104. It was observed that none of the standards were recognised bySC104, suggesting that none of the standards represent the antigen.

Results

The R_(F) values obtained for the standards are shown in Table 6 below.These values are also compared with the range of R_(F) values obtainedfor the variably sialylated SC104 antigen, listed in order of increasingpolarity and degree of sialylation.

TABLE 6 SC104 Standard Structure R_(F) R_(F) Lactosyl G1c-β1,1-Cer 0.79ceramide Globotriaosyl Gal-α1,4Gal-β1,4Glc-β1,1-Cer 0.71 ceramide (Gb3)Globotetraosyl GalNAc-β1,3Gal-α1,4Gal-β1,4Glc-β1,1-Cer 0.62 ceramide(Globoside) GlobopentaosylGalNAc-α1,3GalNAc-β1,3Gal-α1,4Gal-β1,4Glc-β1,1-Cer 0.56 ceramide(Forrsman) GM3 NeuAc-Galβ1,4-Glc-β1,1-Cer 0.53 0.53 0.49 AGM1Galβ1,3-GalNAc-β1,4-Gal β1,4Glc-β1,1-Cer 0.48 GM1Galβ1,3-GalNAc-β1,4-(NeuAc)Gal β1,4Glc-β1,1-Cer 0.39 0.39 0.36 GD3NeuAc-NeuAc-Gal β1,4Glc-β1,1-Cer 0.33 <0.24 GD1aNeuAc-Galβ1,3-GalNAc-β1,4-(NeuAc)Gal β1,4Glc-β1,1-Cer 0.22 GD1bGalβ1,3-GalNAc-β1,4-(NeuAc-NeuAc)Gal β1,4Glc-β1,1-Cer 0.16 GT1bNeuAc-Galβ1,3-GalNAc-β1,4-(NeuAc-NeuAc)Gal β1,4Glc-β1,1-Cer 0.08

It can be seen that the least polar SC104 antigen co-migrates with GM3,while the most polar molecules migrate with R_(F) values similar to GD1aand GD1b. However, in all cases the standards are themselves notrecognised.

The migration of the antigen with R_(F) values that are comparable tomany of the standard gangliosides suggests that, along with therequirement for sialylation, the antigen has a short neutraloligosaccharide backbone that consists of three or four monosaccharides.It is most probable that this is actually a tetraosyl structure as thiswould more readily allow multiple sialylation. The least polar antigenthat is detected is probably a partial antigen structure, consisting ofa shorter oligosaccharide backbone.

Example 6 Identification of Tumour Glycoprotein Recognised by SC104 mabExperiment 1 Methods

SC104 antigen purification and characterisation. SC104 specific antigenwas purified from sputum samples mixed in a 1:1 ratio with 0.1M pH 7.6Tris 0.5% NP40. Briefly an immuno-affinity was produced cross linkingSC104 S136A to Protein A C₁₋₆B sepharose using Dimethylpimelimidate•2HCl as the covalent linker.

Results

It is noteworthy that whilst the antigen was seen to cross-react withSC104 (FIG. 13 a) it did not cross react with an anti-sialyl Lewis^(a)or 19/9 antibody.

Experiment 2 Methods

Sequence identification of the SC104 antigen. SC104 specific antigen waspurified from sputum as outlined in experiment 1 above and the samplesrun on a 10% SDS-PAGE and Silver stained following the suppliersrecommendations (Bio-Rad Silver stain kit, catalogue number 1610443).The band of interest was subsequently subjected to MALDI analysis.

Results

A band for the SC104 antigen at between 50-75 kDa was identified on aSilver stained gel (FIG. 14). The MALDI sequence analysis suggestedseveral possible hits including:

-   -   P04839 GP91-PHOX) (GP91-1) (Heme binding membrane glycoprotein)    -   Q99741 Cell division control protein 6 homolog (CDC6-related        protein)    -   Q14451 Growth factor receptor-bound protein 7 (GRB7 adapter        protein)    -   P14618 Pyruvate kinase, isozymes M1/M2 (EC 2.7.1.40) (Pyruvate        kinase muscle isozyme)    -   O96013 Serine/threonine-protein kinase PAK 4 (EC 2.7.1.37)        (p21-activated kinase 4)    -   P01833 (Polymeric Ig receptor)

As SC104 recognises a carbohydrate, these results suggest that thesialyltetrasoyl sugar can also be expressed on these glycoproteins.

Experiment 3 Methods

Competition assays using the immuno-purified antigen using the SC104protein A sepharose column. The SC104 purified fraction was used as acompetitor for SC104 binding to C170 cells using experiments based uponthe ELISA technique (FIG. 15). Microtitre plates were coated with5×10⁴/well of C170s, the cells fixed and blocked prior to beingincubated with solutions of biotinylated antibody (0.1 μg/well) mixedwith increasing ratio of antigen. Bound SC104 biotinylated antibody wasdetected by SA-HRP and TMB. Results are expressed as absorbance at 650nm.

Results

The immuno-purified SC104 antigen using the protein A C1-6B sepharoseimmuno-affinity column has demonstrated its ability to specificallyinhibit the binding of the antibody to its target.

Example 7 SC104 Directly Induces Tumour Cell Killing Experiment 1Methods

Annexin/PI. Tumour cells (1×10⁵ aliquots) in suspension incubated withSC104 antibody (1 μg/ml) or appropriate controls for 4 hr at roomtemperature could then also be stained with FITC labelled annexin andpropidium iodide (Sigma, Poole, Dorset) and then analysed by dual colourflow cytometry.

Results

This study has shown strong staining of freshly disaggregated colorectaltumours. However during these studies it was observed that SC104antibody appeared to accelerate tumour cell death. A similar phenomenonwas observed when tumour cell lines which normally grew as adherentmonolayers were placed in suspension and stained with SC104 antibodiesfor flow cytometric analysis. To determine if the antibody was inducingapoptosis or necrosis, cells exposed to SC104 antibody werecounterstained with annexin and propidium iodide (FIG. 16). Less than25% of the cells stained with the control antibody showed staining witheither annexin or propidium iodide. In contrast cells exposed toconcentration greater than 10 μg/ml SC104 showed strong staining with30% of cells staining with annexin, 75% with PI and 65% with both. Cellsstained with annexin alone are described as in early stages of apoptosiswhereas cells stained with both annexin and PI are in late stageapoptosis/necrosis.

Experiment 2 Methods

Pan caspase activation. 2×10⁵ colorectal C170 cells were exposed to 30μg/ml of SC104 for up to 6 hrs and pan caspase FITC-FMK-vad (caspACEFITC-vad-FMK, Promega, catalogue number G7462) employed to establishcaspase activation. The cells were analysed by flow cytometry. Controlsincluded a negative control murine antibody and a Fas antibody at 100ng/ml.

Results

The results of the assay demonstrate that SC104 activates pan caspasesafter 5 hrs (FIG. 17).

Experiment 3 Methods

Caspase 6 activation by SC104 mab. 2×10⁵ colorectal C170 cells wereexposed to 1-100 μg/ml of SC104 for 6 hrs and cell lysates generated.The lysates were subsequently run a 12% SDS-PAGE, Western blotted andprobed with a caspase 6 specific antibody (Cleaved caspase 6 antibody,Cell Signalling Technology (NEB), catalogue number 9761). Cells exposedto SC101 were included as a negative control.

Results

Development of the Western blot (FIG. 18) clearly demonstrates thatthere is activation of caspase 6 in those C170 cells exposed to 10, andmore intensely at 100 μg/ml of SC104 mab. Those cells subjected totreatment with the SC101 mab showed no such activation of caspase 6.

Experiment 4 Methods

SC104 inhibition of cell death using the z-FMK-vad caspase inhibitor.2×10⁵ C170 cells were subjected to 3 μg/1 ml of SC104 and half were alsoincubated with 3 μM z-FMK-vad inhibitor (Promega, catalogue numberG7232) and incubated overnight.

The following day cell viability was assayed using annexin V FITC andpropidium iodide.

Results

There was a 23% increase in viable cells in the presence of theinhibitor (FIG. 19), providing a strong indication that SC104 killscolorectal cells by a ‘classic’ apoptotic pathway.

Experiment 5 Methods

Inhibition of cell growth. 1×10³ colorectal C170, C168, C146, CaCO2,Colo205, LoVo and HT29 cells were aliquoted into individual wells of aflat bottomed 96-well plate and left to adhere overnight at 37° C. Thefollowing day the cells were treated with: 10, 3, 1, 0.3 and 0 μg/ml ofSC104 mab. As a negative control 791T/36 at concentrations 100, 30, 10,3 and 0 μg/ml, was titrated against each concentration of drug used.Triplicate wells were used. Cells were left for 5 days at 37° C. priorto the addition of MTS reagent to each well and optical density readingat 490 nm.

Results

The effect of SC104 on adherent cell proliferation was measured in anMTS assay. At concentrations in, excess of 10 μg/ml SC104, but notcontrol antibody, inhibited growth of the colorectal tumour cell lineC170 and Colo205 but not HT29 or LoVo. (FIG. 20). FIG. 21 demonstratesIC₅₀ fits for tumour cell lines C170, Colo205, HT29 and LoVo. Similarresults were obtained with other breast and colorectal tumour cell lines(Table 7). These results suggested that SC104 was inhibiting tumour cellproliferation by inducing apoptosis at high doses. However cells insuspension that had lost their cell/cell contact were sensitized torapid cell death at lower antibody concentrations.

TABLE 7 SC104 inhibits cell growth % inhibition of cell growth asmeasured by MTS staining Cell line SC104 (30 μg/ml) 791T/36 (30 μg/ml)C170 70 10 C146 65 6 C168 55 3 CaCO2 20 2 Colo205 55 5 HT-29 5 5

Experiment 3—Homophilic Binding Methods

FACS on fresh and fixed cells. C170 cells (10⁵) either fresh or fixedwith 0.5% glutaraldehyde for 1 hr were resuspended in 100 μl of SC104mab (0-100 μg/ml) and incubated on ice for 1 hr. After washing thesamples 3 times in RPMI/10% FCS cells were incubated with FITC labelledrabbit anti-mouse ( 1/80: Dako Ltd, Bucks, UK) antibody and incubated onice for 30 minutes prior to analysing by FACS as described above.

ELISA for homophilic binding. Flat flexi 96 well ELISA plates werecoated with anti-mouse IgG Fc specific antibody or were coated with 3μg/ml (100 μg/well) of C14 antigen in PBS and dried down overnight.Plates were washed 3 times with PBS prior to blocking with PBS/1% BSAfor 1 hr at room temperature. SC104 antibody (0-100 μg/ml) was added onice for 1 hr. After washing the plate 3 times in PBS/0.05% tween-20,biotinylated SC104 was added and incubated on ice for 30 minutes.Following a further 3 washes in PBS 0.05% tween-20 anti-mouse SA-HRP (1/1000; Dako, High Wycombe, UK) or goat anti-mouse Fc ( 1/1000: Dako,High Wycombe, UK) was added as appropriate and left on ice for 30 min.Plates were washed 5 times in PBS/0.05% tween-20 before adding 100μl/well of TMB substrate and reading at 570 nm.

Results

The number of SC104 haptens at the surface of a C170 tumour cells isapproximately 4×10⁵ sites per cell. However this number of antigenswould easily be saturated at 1 μg/ml of SC104. It was thereforedifficult to explain why a 10 fold excess of antibody was required forcell killing. It could be that upon antibody binding more sites arerevealed. Antibody saturation curves were therefore generated on freshand fixed cells. The antigen was not saturated even at SC104concentration of 100 μg/ml and curves were similar on fresh and fixedcells making it unlikely that further antigen was being revealed (FIG.22). These results are similar to previously reported data on R24 amouse mab recognising GD3 ganglioside. This antibody shows non-saturableantibody binding and in a series of elegant experiments was shown to bea homophilic binding antibody with the capacity to bind to both antigenand itself (results not shown). SC104 was therefore screened for theability to bind to itself by coating an ELISA plate with unlabelledSC104 and measuring the binding of SC104 biotin HRP-avidin. SC104 didnot bind to itself (FIG. 23) however when purified glycoproteinexpressing SC104 haptens was used to coat the plates SC104 boundstoichiometrically until 10 μg/ml and then bound exponentially beyondthis concentration (FIG. 23). These results suggest that at low antibodyconcentrations the antibody binds to antigen but at high concentrationsantibody bound to antigen can also bind further antibody. This candramatically increase the functional avidity of antibodies leading tolattices of antibodies building up on cells with high density antigen.This lattice formation could result in multiple signals resulting inapoptosis at high antibody concentrations.

Experiment 4—In vitro Inhibition of Cell Growth in Combination withCytotoxic Drugs

1×10³ colorectal C170 cells were aliquoted into individual wells of aflat bottomed 96-well plate and left to adhere overnight at 37° C. Thefollowing day the cells were treated with cisplatin, mitomycin C,oxaliplatin and tamoxifen at final concentrations of 10, 3, 1, 0.3, 0.1and 0 μM. Against each concentration of drug the followingconcentrations of SC104 were titrated: 10, 3, 1, 0.3 and 0 μg/ml. As anegative control 791T/36 at concentrations 100, 30, 10, 3 and 0 μg/ml,was titrated against each concentration of drug used. Duplicate wellswere used. Cells were left for 5 days at 37° C. prior to the addition ofMTS reagent to each well and optical density reading at 490 nm.

Results

The ability of SC104 to show additive killing with chemotherapeuticagents was screened in vitro by MTS assays. All drugs were titratedbetween (0.1-10 μg/ml) in the presence of either SC104 or controlantibody (0.3-10 μg/ml). FIG. 24 shows a representative experiment forcells treated with a combination of SC104 and 5-FU. FIG. 25 summarisesthe results showing that SC104 shows additive killing with cisplatin,mitomycin C, 5-FU and tamoxifen. No additive effect was seen withoxaliplatin.

Experiment 5—In vivo Inhibition of Tumour Cell Growth in Combinationwith Cytotoxic Drugs

Methods

Prevention model. The colorectal tumour cell line, C170 was maintainedin serial passage in nude mice. For therapy the mice were sacrificed andthe tumours excised. The tumour was finely minced and 3 mm² pieces wereimplanted, under anaesthetic, subcutaneously into 30 male mice which hadbeen randomly allocated to 4 experimental groups.

Mice were explanted with 3 mm³ pieces of C170 xenografts. Groups of micewere treated with 5-FU/leucovorin (12.5 mg/Kg) by intravenous infusionon days 1, 3, 5, 7 and 28. On the same days mice were also injectedintraperitonealy with 0.2 mg of SC104 mab. Control mice received eitherSC104 alone or control mouse IgG antibody with 5-FU/leucovorin. Tumoursize was measured by callipers and tumour cross sectional areacalculated on days 7, 9, 12, 14 and 16. Animals were weighed to assessthe toxicity of treatment. At the termination of the experiment tumourswere weighed to assess anti-tumour efficacy.

Therapeutic model. The colorectal tumour cell line, C170 was maintainedin serial passage in nude mice. For therapy the mice were sacrificed andthe tumours excised. The tumour was finely minced and 3 mm² pieces wereimplanted, under anaesthetic, subcutaneously into 30 male mice which hadbeen randomly allocated to 4 experimental groups.

Mice were explanted with 3 mm³ pieces of C170 xenografts. Groups of micewere treated with 5-FU/leucovorin (12.5 mg/Kg) by intravenous infusionon days 3, 5, 7, 21 and 22. On the same days mice were also injectedintraperitonealy with 0.2 mg of SC104 mab. Control mice received eitherSC104 alone or control mouse IgG antibody with 5FU/leucovorin. Tumoursize was measured by callipers and tumour cross sectional areacalculated on days 12, 16, 19 and 23.

Results

To determine if these effects could be translated to inhibition oftumour growth in vivo. SC104 was administered (200 μg/dose) 3 weekly tomice either transplanted with 3 mm² extracts of C170 tumours or to C170tumours that had been allowed to grow for 5 days prior to administrationof the antibody. Animals were treated either with SC104 alone,5-FU/leucovorin at the maximum tolerated dose or a combination of bothfor 3 weeks. FIG. 26 a shows that both SC104 or 5-FU/leucovorin aloneresulted in 50% inhibition of tumour growth of freshly explantedtumours. In contrast, the combination of both showed synergisticinhibition of growth. Tumours in all groups showed a temporal increasein tumour growth until day 16 when the staggered termination began. Fromday 9, the combination treatment group showed significant inhibition ofgrowth when compared with all other treatment groups as follows: —SC104and 5-FU, day 9; 37% inhibition, p=0.004; day 12; 59% inhibition,p=0.002; day 14; 74% inhibition, p=<0.001; day 16; 76% inhibition,p=<0.001. 5-FU, day 9; 40% inhibition, p=0.004; day 12; 45% inhibition,p=0.004; day 14; 58% inhibition, p=<0.001; day 6; 62% inhibition,p=<0.001. SC104, day 9; 32% inhibition, p=0.017; day 12; 44% inhibition,p=0.004; day 14; 54% inhibition, p=0.001; day 16; 62% inhibitionp=<0.001. All statistics assessed by ANOVA.

There was no significant difference between the SC104 treated group andthat receiving the cytotoxic agent, 5-FU/leucovorin. This correlatedinto a significant improvement of survival of mice treated with SC104,5-FU or the combination (FIG. 26 b). The combination of SC104 and 5-FUtreated group showed enhanced survival compared with both the vehiclecontrol group and the 5-FU treated group as follows: —

Comnbination:vehicle: p=0.0038. Combination:cytotoxic: agent p=0.0111(All statistics by Log Rank)

This dose of SC104 was well tolerated with all mice showing no loss ofweight or any other gross pathology (FIG. 26 c). Finally, when SC104 wasadministered therapeutically, 7 days after C170 xenograft tumourimplantation neither 5FU nor SC104 alone significantly inhibited growthhowever in combination with 5FU/leucovorin it both significantlyinhibited tumour growth and enhanced survival (FIG. 27). SC104 plus5-FU/leucovorin exhibited a significant reduction in final weight/timeand inhibited tumour growth with enhanced survival in the C170subcutaneous xenograft model.

1. An isolated specific binding member capable of binding asialyltetraosyl carbohydrate and directly inducing cell death withoutthe need for immune effector cells.
 2. An isolated specific bindingmember capable of binding a sialyltetraosyl carbohydrate, whichsialyltetraosyl carbohydrate is capable of being bound by a membercomprising one or more binding domains selected from domains comprisingan amino acid sequence substantially as set out as residues 44 or 49 to54, 69 to 84 and 117 to 127 of the amino acid sequence of FIG. 1 a.
 3. Abinding member as claimed in claim 2, wherein the binding domaincomprises an amino acid sequence substantially as set out as residues117 to 127 of FIG. 1 a. 4-5. (canceled)
 6. A binding member as claimedin claim 3, further comprising one or both of the binding domainssubstantially as set out as residues 44 or 49 to 54 and residues 69 to84 of the amino acid sequence shown in FIG. 1 a.
 7. A binding member asclaimed in claim 2, wherein each binding domain is carried by a humanantibody framework.
 8. A binding member as claimed in claim 2,comprising the amino acid sequence substantially as set out as residues19 to 138 of the amino acid sequence shown in FIG. 1 a.
 9. A bindingmember as claimed in claim 2, further comprising a human constantregion.
 10. An isolated specific binding member capable of binding asialyltetraosyl carbohydrate, which sialyltetraosyl carbohydrate iscapable of being bound by a member comprising one or more bindingdomains selected from domains comprising an amino acid sequencesubstantially as set out as residues 46 to 55, 71 to 77 and 110 to 118of the amino acid sequence of FIG. 1 c.
 11. A binding member as claimedin claim 10, wherein the binding domain comprises an amino acid sequencesubstantially as set out as residues 110 to 118 of the amino acidsequence of FIG. 1 c. 12-13. (canceled)
 14. A binding member as claimedin claim 11, further comprising one or both of the binding domainssubstantially as set out as residues 46 to 55 and residues 71 to 77 ofthe amino acid sequence shown in FIG. 1 c.
 15. A binding member asclaimed in claim 10, wherein each binding domain is carried by a humanantibody framework.
 16. A binding member as claimed in claim 10,comprising the sequence substantially as set out as residues 23 to 128of the amino acid sequence shown in FIG. 1 c.
 17. A binding member asclaimed in claim 10, further comprising a human constant region.
 18. Aspecific binding member which comprises a binding member as claimed inclaim 2 in combination or association with a binding member as claimedin claim
 10. 19. (canceled)
 20. A pharmaceutical composition comprisinga binding member as claimed in claim 1 and a pharmaceutically acceptableexcipient, diluent, carrier, buffer or stabiliser.
 21. A method for thetreatment of a tumour in a patient which comprises administering to saidpatient an effective amount of a binding member as claimed in claim 1.22. (canceled)
 23. The method of claim 21 further comprisingsimultaneous, separate or sequential administration of an antitumoragent.
 24. The method of claim 23, wherein the antitumor agent isdoxorubicin, taxol, 5-Fluorouracil, irinotecan and/or cisplatin
 25. Anucleic acid encoding a binding member as claimed in claim
 1. 26. Asialyltetraosyl carbohydrate which is capable of being bound by aspecific binding member as claimed in claim
 1. 27. A sialyltetraosylcarbohydrate as claimed in claim 26, further comprising a lipid orprotein backbone.
 28. An isolated polypeptide comprising SEQ ID No. 23.29. An isolated polypeptide comprising one or more domains selected fromthe group consisting of: amino acids 44 to 54 of SEQ ID No. 23; aminoacids 49 to 54 of SEQ ID No. 23; amino acids 69 to 84 of SEQ ID No. 23;amino acids 117 to 127 of SEQ ID No. 23; and combinations thereof. 30.An isolated polypeptide comprising amino acids 44 to 54 of SEQ ID No.23.
 31. An isolated polypeptide comprising amino acids 49 to 54 of SEQID No.
 23. 32. An isolated polypeptide comprising amino acids 69 to 84of SEQ ID No.
 23. 33. An isolated polypeptide comprising amino acids 117to 127 of SEQ ID No.
 23. 34. The isolated peptide of claim 28, whereinthe peptide is capable of inducing cell death with the need for immuneeffector cells.
 35. The isolated peptide of claim 29, wherein thepeptide is capable of inducing cell death with the need for immuneeffector cells.
 36. An isolated polypeptide encoded by a nucleic acidsequence comprising a nucleic acid sequence substantially identical toSEQ ID No.
 24. 37. An isolated polypeptide comprising SEQ ID No.
 27. 38.An isolated polypeptide comprising one or more domains selected from thegroup consisting of: amino acids 46 to 55 of SEQ ID No. 27; amino acids71 to 77 of SEQ ID No. 27; amino acids 110 to 118 of SEQ ID No. 27; andcombinations thereof.
 39. An isolated polypeptide comprising amino acids46 to 55 of SEQ ID No.
 27. 40. An isolated polypeptide comprising aminoacids 71 to 77 of SEQ ID No.
 27. 41. An isolated polypeptide comprisingamino acids 110 to 118 of SEQ ID No.
 27. 42. The isolated peptide ofclaim 37, wherein the peptide is capable of inducing cell death withoutthe need for immune effector cells.
 43. The isolated peptide of claim38, wherein the peptide is capable of inducing cell death without theneed for immune effector cells.
 44. An isolated polypeptide capable ofbinding a sialyltetraosyl carbohydrate, wherein the sialyltetraosylcarbohydrate migrates at one or more of the Rf values of 0.53, 0.49,0.39, and 0.36 when analyzed on a HPTLC plate developed in 50:40:10chloroform:methanol: CaCl₂ (0.5% w/v).
 45. The isolated polypeptide ofclaim 44, wherein the tetraosyl carbohydrate migrates at an Rf value of0.53.
 46. The isolated polypeptide of claim 44, wherein the tetraosylcarbohydrate migrates at an Rf value of 0.49.
 47. The isolatedpolypeptide of claim 44, wherein the tetraosyl carbohydrate migrates atan Rf value of 0.39.
 48. The isolated polypeptide of claim 44, whereinthe tetraosyl carbohydrate migrates at an Rf value of 0.36.
 49. Theisolated peptide of claim 44, wherein the peptide is capable of directlyinducing cell death without the need for immune effector cells.
 50. Anisolated sialyltetraosyl carbohydrate, wherein the tetraosylcarbohydrate migrates at one or more of the Rf values of 0.53, 0.49,0.39, and 0.36 when analyzed on a HPTLC plate developed in 50:40:10chloroform: methanol: CaCl₂ (0.5% w/v).
 51. The isolated sialyltetraosylcarbohydrate of claim 50, wherein the tetraosyl carbohydrate migrates atan Rf value of 0.53.
 52. The isolated sialyltetraosyl carbohydrate ofclaim 50, wherein the tetraosyl carbohydrate migrates at an Rf value of0.49.
 53. The isolated sialyltetraosyl carbohydrate of claim 50, whereinthe tetraosyl carbohydrate migrates at an Rf value of 0.39.
 54. Theisolated sialyltetraosyl carbohydrate of claim 50, wherein the tetraosylcarbohydrate migrates at an Rf value of 0.36.
 55. An isolatedsialyltetraosyl carbohydrate, wherein the sialyltetraosyl carbohydrateis capable of being bound by a polypeptide comprising SEQ ID No.
 23. 56.An isolated sialyltetraosyl carbohydrate, wherein the sialyltetraosylcarbohydrate is capable of being bound by an isolated polypeptidecomprising one or more domains selected from the group consisting of:amino acids 44 to 54 of SEQ ID No. 23; amino acids 49 to 54 of SEQ IDNo. 23; amino acids 69 to 84 of SEQ ID No. 23; amino acids 117 to 127 ofSEQ ID No. 23; and combinations thereof.
 57. An isolated sialyltetraosylcarbohydrate, wherein the sialyltetraosyl carbohydrate is capable ofbeing bound by a polypeptide comprising SEQ ID No.
 27. 58. An isolatedsialyltetraosyl carbohydrate, wherein the sialyltetraosyl carbohydrateis capable of being bound by an isolated polypeptide comprising one ormore domains selected from the group consisting of: amino acids 46 to 55of SEQ ID No. 27; amino acids 71 to 77 of SEQ ID No. 27; amino acids 110to 118 of SEQ ID No. 27; and combinations thereof.
 59. An antigenpreparation comprising a sialyltetraosyl carbohydrate.
 60. The antigenpreparation of claim 59, wherein the sialyltetraosyl carbohydratemigrates at one or more of the Rf values of 0.53, 0.49, 0.39, and 0.36when analyzed on a HPTLC plate developed in 50:40:10chloroform:methanol:CaCl₂ (0.5% w/v).
 61. The antigen preparation ofclaim 60, wherein the sialyltetraosyl carbohydrate migrates at an Rfvalue of 0.53.
 62. The antigen preparation of claim 60, wherein thesialyltetraosyl carbohydrate migrates at an Rf value of 0.49.
 63. Theantigen preparation of claim 60, wherein the sialyltetraosylcarbohydrate migrates at an Rf value of 0.39.
 64. The antigenpreparation of claim 60, wherein the sialyltetraosyl carbohydratemigrates at an Rf value of 0.36.
 65. The antigen preparation of claim59, wherein the sialyltetraosyl carbohydrate is capable of being boundby a polypeptide comprising SEQ ID No.
 66. The antigen preparation ofclaim 59, wherein the sialyltetraosyl carbohydrate is capable of beingbound by an isolated polypeptide comprising one or more domains selectedfrom the group consisting of: amino acids 44 to 54 of SEQ ID No. 23;amino acids 49 to 54 of SEQ ID No. 23; amino acids 69 to 84 of SEQ IDNo. 23; amino acids 117 to 127 of SEQ ID No. 23; and combinationsthereof.
 67. The antigen preparation of claim 59, wherein thesialyltetraosyl carbohydrate is capable of being bound by a polypeptidecomprising SEQ ID No.
 27. 68. The antigen preparation of claim 59,wherein the sialyltetraosyl carbohydrate is capable of being bound by anisolated polypeptide comprising one or more domains selected from thegroup consisting of: amino acids 46 to 55 of SEQ ID No. 27; amino acids71 to 77 of SEQ ID No. 27; amino acids 110 to 118 of SEQ ID No. 27; andcombinations thereof.